In this paper, comparative experiments on the inactivation of Chironomid larvae by ozone and chlorine were conducted. In addition, batch experiments were performed in order to analyze the influence of pH value, organic precursor concentration and temperature on the inactivation efficiency of Chironomid larvae with ozone. Based on it, removal effect of different pre-oxidation combined with coagulation process on Chironomid larvae in raw water was evaluated. The results showed that ozone possessed better inactivation performance than chlorine, and complete inactivation of Chironomid larvae in raw water was obtained at ozone dose of 2.0 mg/L after 25 minutes of contact time. The inactivation rate of Chironomid larvae at pH 6.0 was higher than those found at pH 8.0 in same contact time. Meanwhile, the organic precursor had negative effects on inactivation, indicated by the decreasing inactivation rate from 100% at TOC concentration of 0 mg/L to 20.0% at 8 mg/L after 30 min of contact time. With regard to the temperature, the inactivation efficiency of Chironomid larvae improved with the temperature increasing within the range investigated of 10–25°C. The inactivation rate was reduced by 73.3% when temperature reduced from 25°C to10°C. The coagulation jar test showed that Chironomid larvae in the raw water could be completely removed by ozone pre-oxidation combined with the coagulation process at ozone dosage of 1.2 mg/L.